Vaibhavi Umesh Notebook

From 2007.igem.org

Latest revision as of 00:18, 21 August 2007

My Construction Files
My Sequencing Files

Vaibhaviu 19:02, 1 June 2007 (EDT)

Today we cloned an RFP basic part into pBca9145. So, we did the construction file for pBca9145-Bca1145. We did a PCR of the RFP gene according to the construction file, and the gel is here:

It's a little dilute, but it should work nonetheless. We also digested the vector according to the construction file:

We gel purified the larger of the two bands. It looks like the digest went pretty well, as we see no single-cut plasmid DNA. We took it all the way through to transformation into DH10B cells today.

Vaibhaviu 18:45, 5 June 2007 (EDT)

Today we first observed the transformed plates that grew over the weekend. Four large, white, colonies surrounded by several satellite colonies were observed. No red colonies were observed which meant no parent vector had bled through onto the plate. FOr each colony, we made overnight cultures and placed them in the 'Incushaker'. Chris had already simulated this and two samples of the cultures were already ready for miniprep. We simulated a mini prep experiment and isolated the plasmids from the two culture samples. We then performed the analytical test digest and used BglII and XhoI restriction enzymes. These enzymes were chosen because they gave a distinct digestion pattern when compared with the parent vector.

Then, we simulated a colony PCR on one of the colonies on the plate, chosen randomly. We ran the results of both the digest and the PCR on a gel. The gel contained 5 wells. The first one contained a marker (the ladder), the second one The results of the gel were not very clear. THe parent vector and the PCR lanes both did not show up/ did not display the expected results. THe pictures taken were unclear, but when observed on the UV plate, the bands were brighter, but no additional bands showed up.

We also made the samples to be sequenced. We placed the mini prep plasmids in a 1.5 mL epp tube. We filled out the spreadsheet to send to Quintara and placed the samples in the metal box outside to get picked up. The specific oligos to be used were also mentioned in the spreadsheet.

Vaibhaviu 18:45, 5 June 2007 (EDT)

Today, we got the sequencing results from Quintara

Vaibhaviu 19:02, 12 June 2007 (EDT)

Today I got my sequencing results back. SodC #1 results were not very good and quintara was going to re-send them after sequencing them with a stronger signal. SodC #2 was fully correct. However, at base pair # 549 (on the sequencing results), there was a mis-read of 3 C’s when only two peaks could be seen on the graph. For methAP gene results, methAP#1 yielded a better result than methAP#2. However, although all the base pairs aligned for methAP#1, there was one situation at base pair # 762 (on the sequencing results), where the sequencing result had inserted an extra G not present in the original gene. When the graph was observed, the resulting peak was quite ambiguous and it was difficult to distinguish whether the peak was actually two separate peaks, or was three. As a result, just to double check, the plasmid was sent in for reverse sequencing using the reverse primer G00101 (the reverse primer of ca998). The results for this sequencing file will help establish that the mis-match and presence of an extra G was due to the ambiguity of the graph rather than the presence of an actual mutation.

VU005.MetAP#1 is the sample I sent in along with the primer G00101

Vaibhaviu 19:02, 13 June 2007 (EDT)

Since I had a sample of the SodC gene (#2) which was had perfect sequencing results…I saved that clone on the clonesaver sheet.

Apart from that, today was a really long day because I proceeded to attach ribosome binding sites to the SodC and Map genes I had sequenced. Most of the morning was spent in planning out the procedure as I had to identify the restriction enzymes to use to cut the gene and rbs and attach them together. This was the result of the planning:

RBS: pBca 9145 – Bca1090 SodC gene:

• cut with AlwNI ‡ 1519
• cut with BglII ‡ 1081

9145-Bca1090

• cut with AlwNI ‡ 1538
• cut with BamHI ‡ 553

2nd insertion: RBS: pBca1101 – Bca1128 SodC gene:

• cut with BglI ‡ 923
• cut with BglII ‡ 1677

Bca1128:

• cut with BglI ‡ 1010
• cut with BamHI ‡ 1990

MetAP (map) gene + 1128

MetAP gene:

• cut with BglII ‡ 1950
• cut with BglI ‡ 923

1128

• cut with BamHI ‡ 1990
• cut with BglI ‡ 1010

However, the 1090 rbs miniprep barely had any left in the tube, so I had to transform what was left of the miniprep and grow it out to make some more to use. So I transformed a plate with the 1090 rbs. However,

Vaibhaviu 19:02, 14 June 2007 (EDT)

Today I observed the three plates I had transformed yesterday. There were many colonies on the plate, especially the ones with the ligations on them, making it difficult to isolate individual colonies from the plate. However, I picked three colonies from the plate with the ligations and one colony from the 1090 plate. However, I cannot grow them until 5:00 today because I will not be able to come in before 9 the next day and it is not advisable to exceed 16 hours for growing the colonies. However, as prepartion, the tubes were labeled and the media was poured into them. Now, I examined both the reverse sequencing results for the MetAP #1 gene and the SodC #1 gene which came in. Both the sequences were perfect, without a single error. I saved both these clones on the clonesaver incase I need to use them to make a mini-prep in the future. Since the reverse sequencing results for the MetAP #1 gene were perfect, it can be concluded that the one mismatch error present in the forward sequencing file was a sequencing error rather than an error in the clone.

Since the MetAP #1 gene sequencing results were perfect, I will proceed to attach an rbs to the gene. In order to do this, I need to plan the construction file for the MetAP #1 gene and the 1128- 051/052 rbs plasmids. However, it was found the best sites were those that were cut with BglI and BglII/BamHI, which was the same combination used to digest the rbs site yesterday. As a result, I already had a sample of the rbs site available that had been digested and was ready to use in ligation. So, I went ahead and digested the MetAP #1 gene, ran a gel, extracted the 2kb band and purified, I eluded with 10microliters of water. Then, I ligated the insert along with the MetAP #1 vector bands. After ligation, I transformed the mixture.

One problem encountered was that during the gel purifications step, I made an error and did not label the zymo column I was using to purify the the MetAP #1 gene. I had used another empty zymo column with water to balance it in the centrifuge. Thus, after spinning it once, I no longer knew which zymo column had water, and which held the DNA. Thus, I was forced to continue purification with both zymo columns and ligated the sample from each column with either the 051 insert or 052 insert as I did not have enough of the insert to ligate both samples with both inserts. I also transformed both ligations onto a plate. If all goes well, one of my transformations will NOT yield any colonies and that will be the sample that had contained the water. The other transformation should yield colonies which shows that the bacteria had successfully taken up the plasmid formed during ligation (containing the composite part of the gene+ rbs). The results seen tomorrow will determine the identity of each ligation solution.

Vaibhaviu 19:02, 15 June 2007 (EDT)

I observed the plates I had transformed yesterday. Unfortunately, the plates – one with MetAP #1 and water, and the other with MetAP #1 and and the rbs site both grew colonies which meant my control (using water in the ligation) had failed. I tried trouble shooting by figuring out what went wrong. One possible explanation was that the plasmids had only been singly cut and binded back together during ligation. However, instead of doing a colony PCR to figure out what went wrong, it seemed easier to redo the process. Also, I had to re-do the entire rbs attaching process because I had found out I had used the wrong sequence file to plan the process meaning the restriction enzymes I had used would not work with what I actually had.

I understood the difference between using single rbs’s and rbs libraries. The rbs library had a promoter, and rbs site, and an RFP in the plasmid. However, specific libraries had the promoter replaced with different antibiotic markers. The 51 library had a kanamycin antibiotic marker, while the 52 library had the Cam marker. I also found out that the genes Arthur and I had been working on needed to be paired as they performed a similar function. I was planning on attaching a single rbs and an rbs library to each gene that I was using.

Single strong rbs sites I could use:

• 1090
• 1106A
• 1106B
• 1128A

The rbs libraries I could use:

• pBca1101 – I716051 (kanamycin resistance)
• pBca1101 – I716052 (cam resistance)

The genes would be paired in the following way:

• KatG + SodC ‡ hydrogen peroxide
• Map + AHSP ‡ hemoglobin

AHSP was the synthesized gene that austin had cloned into Bca9145. Today I focused on Map + AHSP and planned out how I would attach rbs sites to these genes.

Map gene + 1090

Map gene + library (51)

Map gene: 51 (rbs library) BglII – 2864 BamHI - 2904 EcoRI – 9 EcoRI - 1065

AHSP + 1090

AHSP gene: 1090 (rbs) AlwNI – 1519 AlwNI - 1538 BglII - 868 BamHI - 553

AHSP + library (52)

AHSP gene: 52 (rbs library) BglII – 2378 BamHI - 2904 EcoRI – 9 EcoRI - 933

I had to leave a little early due to a dance rehearsal that had been scheduled so this was as far as I got today.

Vaibhaviu 19:02, 18 June 2007 (EDT)

Today I started the process of attaching rbs sites to the Map + AHSP genes I had planned out on Friday. I followed the plan and digested the genes, ran a gel and extracted the bands. I gel purified and put the sample in the freezer. I would finish the process tomorrow.

Vaibhaviu 19:02, 19 June 2007 (EDT)

Today I continued to ligate and transform the stuff I had started yesterday. After I finished transforming the plates, Arthur gave me the katG gene in the 9415 plasmid so I will first plan out the digestion for both the katG gene and the sodC gene, perform a restiction digest, run a gel, and extract the bands and gel purify today. That way, I can come in tomorrow and ligate and transform. both the katG gene and the sodC gene.

SodC + 1090 SodC: Digest with AlwNI: 1519 Digest with BglII: 1081

1090: Digest with AlwNI: 1538 Digest with BamHI: 553

SodC + library (51)

SodC: Digest with EcoRI:9 Digest with BglII: 2591

51 (library): Digest with EcoRI: 1065 Digest with BamHI: 2904

KatG + 1090 ‡ 1-2-3 method

KatG: Digest with: Digest with BglII:

1090: Digest with: Digest with BamHI:

KatG + library (52)

KatG: Digest with EcoRI: 9 Digest with BglII: 4250

52 (library): Digest with EcoRI: 933 Digest with BamHI: 2904

• Because there were many internal restriction sites for the katG gene making it difficult to attach the 1090 rbs by normal methods, I had to use the 1-2-3 method. I read about this procedure on the arkin wiki.

Vaibhaviu 19:02, 20 June 2007 (EDT)

Today I started the first step of the 1-2-3 method to attach the 1090 rbs to the katG gene. I had to transform the katG gene miniprep into the righty strain and the rbs (1090) miniprep into the lefty strain. I plated both of these. I also digested and gel purified the following SodC & 1090 KatG & 52 SodC & 51

Vaibhaviu 19:02, 21 June 2007 (EDT)

Today I continued what I was doing yesterday…tying up a lot of loose strings! I had transformed the lefty and righty plates yesterday. I got colonies, however, they were not very uniformly spread out..which might have been a result of the way I had spread the culture on the plate. I was able to isolate two colonies on each plate and I grew them in the incushaker. Then, the plasmids I had gel purified yesterday needed to be ligated. I ligated the fragments and proceeded to transform them.

• SodC + 1090 was plated on an Amp plate
• KatG + 52 was first grown out a little with LB Agar in the incushaker for an hour
  • It was then plated on an Amp + Cam plate
• SodC + 51 was first grown out a little with LB Agar in the incushaker for an hour
  • It was then plated on an Amp + Kan plate

Vaibhaviu 19:02, 22 June 2007 (EDT)

LAB FLOODED…no work done!

Vaibhaviu 19:02, 25 June 2007 (EDT)

• Grew colonies from plates transformed on Wednesday
  • SodC + 1090 (single rbs)
  • SodC + 51 (rbs library)
  • KatG, HPI + 52 (rbs library)
• For katG, HPI + 1090 (rbs single) however, due to the presence of numerous internal restriction sites, in order to compose the composite part, Chris’s 1-2-3 method was employed
• The colonies that were transformed – part of the lefty and righty parts were grown and ready on Friday
• Due to the lab flooding…no work was done on Friday
• Two colonies were picked and grown for each plate, so I had 2 colonies from the lefty plate and two from the righty plate
• I mini prepped these samples and ran the 1-2-3 program on them
• As the last step of the 1-2-3 program, I transformed the plasmid mixture into a righty strain of the bacteria.

Vaibhaviu 19:02, 26 June 2007 (EDT)

• I took out the plate from the incubator..this plate was the katG + 1090 which I had made via 1-2-3.
• I grew colonies from this plate
• Also, I found out that the libraries of rbs’s that I had grown on a plate had to be grown differently than just picking a few colonies and growing them
• Since I had already done this…I re-did those plates…the sodC + 51 and katG + 52 and grew them by adding LB and making a homogenous mixture.
• I then took a microliter of the homogenous mixture and put it into the tube containing the media
• However, the SodC + 1090 was ready to be miniprepped so I miniprepped that
• Along with that, I also sent in the following for sequencing

What I sent in for sequencing: VU006 – map + 1090 #1 VU007 – map + 1090 #2 VU008 – AHSP + 1090 #1 VU009 – AHSP + 1090 #2 VU010 – SodC + 1090 #1 VU011 – SodC + 1090 #2

• I spent a lot of time organizing my box and sorting through all my mini-preps and stuff and categorizing everything

Tomorrow’s plan:

• Grow the map + 51 and the AHSP + 52 Transform katG + 52 into righty
• Transform SodC + 1090 and SodC + 51 into lefty
• Miniprep SodC + 51, katG + 52 and katG + 1090
• Send in the katG + 1090 for sequencing
• Analyze sequencing results

Vaibhaviu 19:02, 27 June 2007 (EDT)

• I miniprepped…
  • katG + 52
  • SodC + 51
  • KatG + 1090 (#1)
  • KatG + 1090 (#2)
• Sent in katG + 1090 (#1) and katG + 1090 (#2) for sequencing
• Transformed katG + 52 into righty
• Transformed SodC + 1090 and SodC + 51 into lefty

Gene + rbs Where it is What needs to be done immediately Work for the future Map + 1090 Miniprepped (3 samples) Sequencing Transformed into lefty strain Map + 51 AHSP + 1090 Miniprepped (3 samples Sequencing Transformed into righty strain AHSP + 52 SodC + 1090 Miniprepped (2 samples) Sequence needs to be analyzed Needs to be transformed into lefty SodC + 51 Miniprepped ( 1 sample…CORRECT) KatG + 1090 Miniprepped (2 samples) Sequencing Transformed into righty strain KatG + 52 Miniprepped (1 sample…CORRECT)

What I sent in for sequencing:

VU012 – katG + 1090 #1 VU013 – katG + 1090 #2 VU014 – map + 1090 #1 VU015 – map + 1090 #2 VU016 – AHSP + 1090 #1 VU017 – AHSp + 1090 #2

• Essentially I have two operons – both of them contain the same genes in the same order, however, one system has the genes attached to a single rbs site, while the other system has the genes attached to an rbs library.
• The advantage of doing both of these things simultaneously is that in case the single rbs doesn’t work and I need to try to find a stronger one within the library, I will not waste time by starting to add in the libraries to the genes and making the gene + rbs cassette then.
• For the single rbs sites attached to the genes I will use the 1-2-3 method to assemble the cassettes together in the following way:

LEFTY RIGHTY Map + 1090 AHSP + 1090 SodC + 1090 KatG + 1090

• For the rbs libraries attached to the genes, I will do an EcoRI/BamHI and EcoRI/BglII digest to attach the two cassettes together

Vaibhaviu 20:08, 28 June 2007 (EDT)

• KatG + 1090 ‡ analyze sequencing results
• NO GENEEEE
• Colony PCR will be performed to test whether the gene has been cloned into any of the colonies
• COLONY PCR
  • Water: 21.8 x 12 = 261.6 microliters
  • Thermol pol buffer 2.5 x 12 = 30 microliters
  • DNTPs (10mM) 0.5 x 12 = 6 microliters
  • Taq 0.1 x 12 = 1.2 microliters
  • Forward primer 0.5 x 12 = 6 microliters
  • Reverse primer 0.5 x 12 = 6 microliters

I put the PCR tubes in the machine and also poured media into all the tubes simultaneously so that I can grow the cultures while the PCR is going

The PCR is done…I am going to load gels and observe the bands seen to test whether the PCR worked.

• I saved the clones from yesterday. I took 3 microliters of plasmid and added it to 10 microliters of water and cloned saved all 13 microliters

Save the following clones: SodC + 1090 ‡ VU010 is the good one (#1) Map + 1090 ‡ VU006 is the good one (#1)

• I also transformed a bunch of stuff into lefty and righty parts so that I could make composite parts as I needed
• This is the stuff I transformed:

SodC + 1090 lefty and righty Map + 1090 lefty and righty AHSP - righty 1090 – lefty

• Since my sequencing results for the AHSP + 1090 showed that my cloning had failed… I decided to re-try making the composite part with the 1-2-3 method which is why I transformed them into the righty and lefty parts respectively.

• After I ran the gel and took a picture, only one of the colonies picked had a band…#9…which means that I can mini-prep this and get it sequenced and potentially have the right result.

• These are things I have to re-do. I cannot use 1-2-3 method for the rbs libraries attached to the genes so I have to use the older method.

Things to re-do Map + 51 AHSP + 52 AHSP + 1090 ‡ (1-2-3 method)

Vaibhaviu 17:07, 29 June 2007 (EDT)

• Miniprepped Sample 9 and the Control
• Sent both in for sequencing
  • VU014: Colony PCR (katG + 1090) #9
  • VU015: katG righty (CONTROL)
• I began the process of re-doing the gene + library. However, when I was about to let it digest, I found that I did not have enough of the Map gene to complete the process. So, I transformed both the AHSP and Map gene so that I could grow it and miniprep it and complete the process on Monday.

Apart from that I just updated the notebook and will try to continue organizing the miniprep box.

Vaibhaviu 19:53, 2 July 2007 (EDT)

• Today I miniprepped 12 samples…they were the sodC + 1090 and map + 1090 transformed them into lefty and righty strains. That was 8 samples.
• In addition, I transformed two samples of AHSP into righty and two samples of 1090 rbs into lefty.
• I proceeded to perform 1-2-3 on the AHSP + 1090. I will transform the mixture and come back tomorrow to grow it and then I will send it in for sequencing. Hopefully it will work
• I transformed the mixture into DH10B, Lefty, and Righty strains
• I got back the sequencing results for the katG + 1090 on which I had performed a colony PCR. Turns out that I only got the gene and the rbs had not been attached. This seems to be a problem with the 1-2-3 method. You end up getting one part or the other, rarely both.
• Tomorrow, I will try to perform 1-2-3 on the mixture agai
• Also, I grew out the colonies I had transformed of the AHSP and map gene as I did not have enough to do a digest to attach the 51/52 libraries to them.
• The plates only had 5-6 large colonies, which was strange as I expected a lot more. However, since I had transformed plasmids, it is unnecessary to send it in for sequencing since I was just making more of the miniprep.

I grew out two colonies from each of these plates, which I will miniprep tomorrow

Tomorrow’s plan

• Miniprep the two cultures and proceed to do a restriction digest and extract and purify, etc….to attach the rbs libraries to the gene
• Grow cultures from the plates transformed today
• Organize mini-prep box and clone save the necessary
• Find out if Austin needs the AHSP gene + rbs library in DH10B or in a lefty/righty strain and transform it accordingly

4Th OF JULYYYYYY!!! I came into lab for only some time today. I was here for about 2.5 hours. I just ligated and transformed the stuff I had gel purified yesterday.

1. Map gene 1 + 51 2. Map gene 2 + 51 3. AHSP gene 1 + 52 4. AHSP gene 2 + 52 5. AHSP (Austin) + 52 6. Map gene 1 + 51 (test with ligase buffer that was kept outside and needed to be tested for its productivity) 7. Map gene 2 + 51 (test with ligase buffer that was kept outside and needed to be tested for its productivity) Before plating however, the transformation mixture needed to be grown. The samples needed to be grown for an hour in media since they contained Kan and Cam resistance.

Vaibhaviu 18:34, 5 July 2007 (EDT)

• Today I came in and looked at the plates that had been transformed yesterday.
• Most of the plates had lots of colonies on them, however, I needed to make a homogenous mixture of the colonies by adding 2mL of LB media
• I pippetted out 1 microliter of this mixture and put it into the respective tube filled with either Kan + Amp media or Cam + Amp media based on whether the sample had

a 51 or 52 rbs library

• I also finished the colony PCR started earlier in the week. I added loading buffer to all the 32 PCR samples and ran the gels.
• The results were as follows:
  • 1(1) – worked – sent for sequencing (VU016)
  • 1(2) - failed
  • 1(3) – worked – sent for sequencing (VU017)
  • 1(4) – worked – sent for sequencing (VU018)
  • 1(5) - failed
  • 2(1) – worked – sent for sequencing (VU019)
  • 2(2) – worked – sent for sequencing (VU020)
  • 2(3) – worked – sent for sequencing (VU021)
  • 2(4) - failed
  • 2(5) - failed
  • 3(1) – worked – sent for sequencing (VU022)
  • 3(2) - failed
  • 3(3) – worked – sent for sequencing (VU023)
  • 3(4) – worked – sent for sequencing (VU024)
  • 3(5) - worked
  • 3(6) - worked
  • 4(1) – worked – sent for sequencing (VU025)
  • 4(2) – worked – sent for sequencing (VU026)
  • 4(3) – worked – sent for sequencing (VU027)
  • 4(4) - worked
  • 4(5) - worked
  • 4(6) - failed
  • 5(1) – worked – sent for sequencing (VU028)
  • 5(2) - failed
  • 5(3) - failed
  • 5(4) - failed
  • 5(5) – worked – sent for sequencing (VU029)
  • 6(1) – worked – sent for sequencing (VU030)
  • 6(2) – worked – sent for sequencing (VU031)
  • 6(3) – worked – sent for sequencing (VU032)
  • 6(4) - worked
  • 6(5) – CONTROL - worked

• All the cultures that worked will be grown out and miniprepped tomorrow. I have 30 minipreps to do tomorrow, so I will label all the tubes required beforehand so that I can come in early tomorrow and get started

Vaibhaviu 17:34, 6 July 2007 (EDT)

• I miniprepped all 30 samples. I used ‘pig’ the vacuum miniprep device. It took FOREVER!!!!
• I sent some of the samples off for sequencing
• I proceeded to try to attach a 1090 rbs to the katG gene. I learnt about the DBBS method. I planned out the protocol for this method
• I prepared the PCR, which was the first step of the process and left it overnight. I was going to return the next morning and purify and transform.

Vaibhaviu 9:34, 7 July 2007 (EDT)

• I came in this morning and ran a gel of the PCR product and purified simultaneously.
• After I finished purifying, I examined the results.
• Unfortunately, the picture showed that no bands showed up, meaning the PCR had not worked.

Vaibhaviu 20:27, 9 July 2007 (EDT)

• I analyzed the results I had gotten. The results were as follows:

• • 1(1) – worked – sent for sequencing (VU016) – results: only gene, no rbs
  • 1(2) - failed
  • 1(3) – worked – sent for sequencing (VU017) - results: only gene, no rbs
  • 1(4) – worked
  • 1(5) - failed
  • 2(1) – worked – sent for sequencing (VU018) - results: gene + rbs -- SUCCESS!!!! (DH10B (AHSP(2) + 1090)
  • 2(2) – worked – sent for sequencing (VU019) - results: only rbs, no gene
  • 2(3) – worked
  • 2(4) - failed
  • 2(5) - failed
  • 3(1) – worked – sent for sequencing (VU020) - results: gene + rbs -- SUCCESS!!!! (Lefty (AHSP(1) + 1090)
  • 3(2) - failed
  • 3(3) – worked – sent for sequencing (VU021) - results: only gene, no rbs
  • 3(4) – worked
  • 3(5) - worked
  • 3(6) - worked
  • 4(1) – worked – sent for sequencing (VU022) - results: only gene, no rbs
  • 4(2) – worked – sent for sequencing (VU023) - results: only gene, no rbs
  • 4(3) – worked –
  • 4(4) - worked
  • 4(5) - worked
  • 4(6) - failed
  • 5(1) – worked – sent for sequencing (VU024) - results: only gene, no rbs
  • 5(2) - failed
  • 5(3) - failed
  • 5(4) - failed
  • 5(5) – worked – sent for sequencing (VU025) - results: gene + rbs -- SUCCESS!!!! (Righty – AHSP (2) + 1090)
  • 6(1) – worked – sent for sequencing (VU026) - results: gene + rbs -- SUCCESS!!!!

(Righty – AHSP (2) + 1090)

• • 6(2) – worked – sent for sequencing (VU027) - results: gene + rbs -- SUCCESS!!!!

(Righty – AHSP (2) + 1090)

• • 6(3) – worked –
  • 6(4) - worked
  • 6(5) – CONTROL – worked - sent for sequencing (VU028) – results: matches up (does not contain the A of the AGATCT site in the beginning)

• Since I had samples that worked, I proceeded on re-doing the DBBS method on the katG + 1090 gene.
• I prepared a PCR and waited for it to complete and purified it
• I ran the purified PCR product on a gel to see if the PCR worked and the rbs worked, but the gene barely had a band visible. I thought I saw a faint band for the gene, so I continued with the digestion, but set up another PCR for the gene on the side which I will purify and digest tomorrow if I don’t get any colonies tonight.
• Did a restriction digest using BsaI and BglII for Part B (katG gene) and BsaI and BamHI for Part A (1090 single rbs).
• Purified the restriction digest
• Ligated
• Transformed in DH10B

Vaibhaviu 18:10, 10 July 2007 (EDT)

• I got COLONIES for the dBBS method plate!!!! Since I used dpnI to get rid of the parent vector, the colonies that grew should be the correct clone of the gene + rbs.

I will send it in for sequencing to check.

• Today I was going to plan out how to make composite parts of the genes + rbs’s that I have.
• This is what I have so far:
  • 1090 rbs + SodC gene
  • 51 rbs library + SodC gene
  • 1090 rbs + katG gene --- need to send for sequencing
  • 52 rbs library + katG gene
  • 1090 rbs + map gene
  • 51 rbs library + map gene
  • 1090 rbs + AHSP gene
  • 52 rbs library + AHSP gene

• For composite parts, these are the pairs that I will make
  • (1090 + SodC) + (1090 + katG)
  • (51 + SodC) + (52 + katG)
  • (1090 + map) --- put in front of promoter (T7)
  • (51 library + map) --- put in front of promoter (T7)
  • (1090 + AHSP) --- coordinate with Austin’s system
  • (52 + AHSP) --- coordinate with Austin’s system

• I can work on everything except the first composite part set because I have not sent the sample (1090 + katG) in for sequencing.
• I organized and categorized all my miniprep boxes and threw out samples that did not work so that I could easily find the samples I needed

• Plan for the week:

• • TUESDAY
    • grow colonies from the katG + 1090 plate
    • find out how to deal with (51 library + map) + promoter (can you transform them into lefty/righty)?
    • update all construction files + parts into the registry
    • plan out exactly what you plan to do with exact number of bp of the parts needed etc for tomorrow

• • WEDNESDAY
    • digest the samples:(51 + SodC) + (52 + katG)
    • digest the samples: (map + 1090) and the T7 promoter
    • gel extract and purify everything
    • ligate and transform all samples
    • mini prep and send the katG + 1090 sample for sequencing

• • THURSDAY
    • analyze katG + 1090 sample – if it works,
    • digest katG + 1090 and sodC + 1090
    • gel extract and purify everything
    • ligate sample and transform it
    • grow out all the colonies transformed on wednesday

• • FRIDAY
    • miniprep samples grown on Wednesday and send some for sequencing
    • grow out the [katG + 1090] + [sodC + 1090]

• • SATURDAY
    • miniprep sample grown out on Friday and send for sequencing

Vaibhaviu 21:08, 11 July 2007 (EDT)

• I digested the samples required for the composite parts according to the construction files
• While I was waiting for my digestion, I miniprepped the three colonies I picked yesterday from the katG + 1090 plate (dBBS method). I will send them to be sequenced today so that I will know whether my clones worked or not.
• Sequencing samples:
  • VU029: katG + 1090 (dBBS method) 1
  • VU030: katG + 1090 (dBBS method) 2
  • VU031: katG + 1090 (dBBS method) 3

• I finished up the gel extraction, ligation, and transformation for the composite parts.

Vaibhaviu 21:08, 12 July 2007 (EDT)

• I got sequencing results back…and NONE of them worked. I got only the rbs part
• I need to re-do colony pcr on the plate. to test for positive results and based on that send more in for re-sequencing
• I will perform colony pcr tomorrow so that when I grow my cells tomorrow, I can come in on Saturday and miniprep them
• I can miniprep my composite part tomorrow
• If I transform today, I can grow everything out tomorrow/colony pcr
• If I transform tomorrow, then I can come in and grow things out on Saturday

Vaibhaviu 21:08, 13 July 2007 (EDT)

• I re-did the colony pcr on the katG + 1090 plate. The program would take 4-5 hours, so I would leave it overnight and come in on Saturday to run the gel on them.
• I mini-prepped

Vaibhaviu 21:08, 14 July 2007 (EDT)

• I came in and ligated the T7 +{[katG + 52] + [sodC + 51]}part and transformed it into DH10B
• I also performed the colony pcr on the 10 colonies I had done pcr on the night before from the katG + 1090 plate (dBBS method)
• I ran the pcr products on a gel and all the colonies I picked tested positive, which means that either the colonies are successful clones of the gene + rbs, or contain only the gene. I will miniprep them all on Monday and send in 3-4 samples for sequencing and hope they are successful clones!

Vaibhaviu 21:08, 16 July 2007 (EDT)

• I miniprepped 15 samples! It took a while..because the centrifuge broke the tops off my eppendorf tubes so I had re-label everything!
• I will send all the relevant samples off for sequencing
• I will also digest and gel extract AHSP + 51 and AHSP + 1090 parts so I can combine both these parts with promoters and give the completed cassette (with a terminator as well) to Austin so he can assay it with his system
• Sequencing Stuff Results:
  • VU032 – dBBS method (katG + 1090) #1
  • VU033 – dBBS method (katG + 1090) #2
  • VU034 – dBBS method (katG + 1090) #3
  • VU035 – dBBS method (katG + 1090) #4
  • VU036 – T7 + [1090 +map] #1
  • VU037 – T7 + [1090 +map] #2
  • VU038 – T7 + [1090 +map] #3

Vaibhaviu 21:08, 17 July 2007 (EDT)

• I ligated and transformed [AHSP + (1090)] + T7 and [AHSP + 52 library] + T7
• Sequencing Stuff Results:
  • VU032 – dBBS method (katG + 1090) #1 - FAILED
  • VU033 – dBBS method (katG + 1090) #2 - FAILED
  • VU034 – dBBS method (katG + 1090) #3 - FAILED
  • VU035 – dBBS method (katG + 1090) #4 - FAILED
  • VU036 – T7 + [1090 +map] #1 - SUCCESS
  • VU037 – T7 + [1090 +map] #2 - SUCCESS
  • VU038 – T7 + [1090 +map] #3 – SUCCESS
• I had also transformed VU036 – VU038 and also {[katG + 52] + [sodC + 51]}+ T7 promoter into the BLR strains so that the gene expression could be monitered. So, from these plates, I had sufficient colonies for the katG + sodC plate, and VU036 and VU038. I grew all the colonies from the katG + sodC plate (since it was a library) and picked two colonies from VU036 and VU038 and grew those as well.
• The plates that did not grow were VU037 and map + 52 + T7 plate.
• Since the sequencing failed, I re-did the dBBS method on the katG + 1090 composite part
• I pcr-ed the product and pcr purfied it.

Vaibhaviu 21:08, 18 July 2007 (EDT)

• I conintued with the dBBS method. I digested the product and will gel purify it before the baseball game.
• I grew colonies from the plates I had transformed yesterday. These were the following plates I had transformed.
  • map + 52 + T7 plate ‡ good colonies ‡ grew this out
  • 3(1) (AHSP + 1090 + T7)
  • 2(1) (AHSP + 1090 + T7) ‡ not enough colonies
  • AHSP (Austin) + 52 + T7 ‡ not enough colonies
  • AHSP (1) + 52 + T7 ‡ good colonies ‡ grew this out
  • AHSP (2) + 52 + T7 ‡ good colonies ‡ grew this out
  • map + 51 + T7 in BLR ‡ not enough colonies

Vaibhaviu 21:08, 20 July 2007 (EDT)

• the various pcr mixtures ALL FAILED!!!!
• one of them had a faint band so I digested that one and gel extracted, and gel purified it
• I ligated this with the 1090 one I had purified two days ago on Wednesday.
• I also decided to transform the relevant cassettes into the BLR strain (those cloned with a T7 promoter)
• I also sent in various samples in for sequencing.
• samples sent in for sequencing include:
  • VU039 – (sent in to campus) katG sample that Arthur had originally given me to check the gene is in good shape
  • VU040 – [3(1) – AHSP + 1090 + T7 #1]
  • VU041 – [3(1) – AHSP + 1090 + T7 #2]
  • VU042 – [2(1) – AHSP + 1090 + T7 #1]
  • VU043 – [2(1) – AHSP + 1090 + T7 #2]

Vaibhaviu 21:08, 22 July 2007 (EDT)

• I came in today and grew out a few colonies
  • AHSP (1) + 52 lib + T7 promoter in BLR
  • AHSP (2) + 52 lib + T7 promoter in BLR
  • AHSP (2(1) #1) + 1090 + T7 in BLR‡ two colonies
  • AHSP (3(1) #1) + 1090 + T7 in BLR ‡ two colonies

planned agenda for 7/23/07

• miniprep required samples grown yesterday
  • AHSP + 52 + T7 (both samples)
• transform colonies from 2(1) #2 into BLR
• start cloning to form sodC + T7 and katG +T7
• colony pcr for the dBBS method to see what worked ‡ pick 10 colonies
• in spare time: write out assay that will be performed step by step. Look up assays for map gene
• Sequencing results:
  • VU040 – [3(1) – AHSP + 1090 + T7 #1] ‡ failed
  • VU041 – [3(1) – AHSP + 1090 + T7 #2] ‡ failed
  • VU042 – [2(1) – AHSP + 1090 + T7 #1] ‡ failed
  • VU043 – [2(1) – AHSP + 1090 + T7 #2] ‡ successful

Vaibhaviu 19:02, 23 July 2007 (EDT)

• some deviations were made from the planned agenda.
• I don’t have to miniprep BLR samples as I need the culture to assay. So, I kept those cultures in the fridge and will transform the required plasmids into BLR when I am ready to assay..probably by Wednesday or Thursday
• I did a colony pcr on the dBBS plate (katG + 1090)
• I picked 10 colonies and here are the results: ‡ they all failed!
• I got the katG gene re-sequenced to check that the plasmid is still intact.
• VU044 – katG gene
• I also cloned the following so I can assay the genes
  • 52 rbs library + katG gene + T7 promoter
  • 51 rbs library + sodC gene + T7 promoter
  • 1090 rbs + sodC gene + T7 promoter

Vaibhaviu 14:52, 13 August 2007 (EDT)

• Today I did not have much work.
• I analyzed sequencing results:
  • VU057: FAILED
  • VU058: FAILED
  • VU059: FAILED
  • VU060: FAILED
  • VU061: FAILED

• I sent in a few more samples of AHSP + 1090 + t7 + Terminator minipreps into sequencing
  • VU062: (AHSP + 1090 + T7 + Terminator) Sample 6
  • VU063: (AHSP + 1090 + T7 + Terminator) Sample 7
  • VU064: (AHSP + 1090 + T7 + Terminator) Sample 8
  • VU065: (AHSP + 1090 + T7 + Terminator) Sample 9
  • VU066: (AHSP + 1090 + T7 + Terminator) Sample 10

• I troubleshot for the failed cloning of the [AHSP + 1090 + T7] + Terminator
• I decided to transform them into Lefty and Righty strains today so that I can miniprep them and clone on Wednesday.
• Stuff transformed:
  • [AHSP + 1090 + T7] – Lefty strain
  • [AHSP + 52 + T7] – Lefty strain
  • [Terminator sequence] – Righty strain

• I also grew out my glycerol stocks in LB + Amp media
• I grew my sodC constructs out in minimal media
• In case my cultures are too old: transform the stuff that I need
  • SodC + 1090 in BLR
  • katG + 52 in BLR
  • SodC + 51 in BLR
  • 51 + katG + 52 + sodC in BLR
• Just to make sure my minimal media is working, I will run a trail run as well in a 96 well plate with the minimal media with an old LB + Amp culture.

Time course planned for the week for acclimatizing cells to minimal media:

• Tuesday:

Pick colonies from the plates and grow them in 96 well plates – you should have at least 3 plates, if not 4 (do it by 10 am)

• Wednesday:

Take the colonies and put them into minimal media and grow for 30 hours (9 am)

• Thursday:

Put into second round of minimal media (3 pm) OR let them grow until (8 pm)

• Friday

Put into third round of minimal media – late

• Saturday:

• Sunday:

Tecan: do the paraquat and H2O2 assays and put it in for 10 hours

Vaibhaviu 16:59, 14 August 2007 (EDT)

• Today I analyzed the seqencing results.
  • VU062: SUCCESSFUL – except for a point mutation ‡ the point mutation is not a silent mutation
  • VU063:FAILED
  • VU064: FAILED
  • VU065: SUCCESSFUL – except for a point mutation ‡ the point mutation is the same, but is not a silent mutation.
  • VU066: FAILED
• I also grew out the lefty and righty plates which I transformed. The plasmid transformation was super efficient and so I got a lawn. So, it was difficult to pick one colony from the plate. As a result, I re-transformed the lefty and righty strains in case the culture tubes did not grow.
• I will pick colonies for the three 96 well plates
  • SodC + 1090 and SodC + 51 in one 96 well plate
  • KatG + 52 in one 96 well plate
  • KatG + 52 + SodC + 51 in one 96 well plate

Vaibhaviu 20:18, 20 August 2007 (EDT)

• Today I did a variety of things
• I miniprepped 8 samples
  • T7 + 52 (1) + AHSP + 1102-1124 (individually picked colony 1)
  • T7 + 52 (1) + AHSP + 1102-1124 (individually picked colony 2)
  • T7 + 1090 + AHSP + 1102-1124 #1
  • T7 + 1090 + AHSP + 1102-1124 #2
  • T7 + 1090 + AHSP + 1102-1124 #3
  • T7 + 1090 + AHSP + 1102-1124 #4
  • T7 + 1090 + AHSP + 1102-1124 #5 ‡ older culture (grown for 2 days so 6mL of culture was miniprepped)
  • T7 + 52 (2) + AHSP + 1102-1124 (library) ‡ older culture (grown for 2 days so 6mL of culture was miniprepped)

I sent in the following for sequencing:

• • VU067: T7 + 52 (1) + AHSP + 1102-1124 (individually picked colony 1)
  • VU068: T7 + 52 (1) + AHSP + 1102-1124 (individually picked colony 2)
  • VU069: T7 + 1090 + AHSP + 1102-1124 #1
  • VU070: T7 + 1090 + AHSP + 1102-1124 #2
  • VU071: T7 + 1090 + AHSP + 1102-1124 #3
  • VU072: T7 + 1090 + AHSP + 1102-1124 #4
  • VU073: T7 + 1090 + AHSP + 1102-1124 #5

• I also had to put the cultures into Minimal Media round 3
• Here are OD measurements made earlier on Minimal Media round 1
• Here are the OD measurements made on Minimal Media round 2:
• Absorbance measured, Parameters = 600 nm, K = 1.0000
  • SodC + 51 plate
    • A1: 0.065
    • A2: 0.019
    • B1: 0.063
    • C1: 0.269
    • C4: 0.008
  • katG + 51 plate
    • A1: 0.049
    • A2: 0.010
    • B1: 0.015
    • C1: 0.074
    • C4: 0.041

• I also learnt how to make LB broth and LB Agar and plates of those two.
• I went through this entire process and made plates so I could transform the colonies I needed to sent my parts into the registry.
• Colonies plated and will grow:
  • (1) – SodC gene #2 ‡ product:(I716)
  • (2) – MetAP #1 ‡ product:(I716)
  • (3) – katG gene ‡ product:(I716253)
  • (4) – AHSP gene (1) ‡ product:(I716)
  • (5) – map + 1090 (1) ‡ product:(I716)
  • (6) – SodC + 1090 (1) ‡ product:(I716)
  • (7) – 2(1) – AHSP + 1090 ‡ product:(I716)
  • (8) – map + 1090 + T7 (1) ‡ product:(I716)
  • (9) – sodC + 1090 + T7 (1) ‡ product:(I716)
  • (10) – 2(1) AHSP + 1090 + T7 #2 ‡ product:(I716)

• Based on the time, I might make glycerol stocks of the cultures grown in LB + Amp media. Otherwise, it will the first thing I do when I come in tomorrow.
• I also have to look at Tecan availability so I can run my plates. Most likely, I will plan it out tonight and make the required solutions tomorrow and run the tecan Wednesday.